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Endogenous capsid-forming protein ARC for self-assembling nanoparticle vaccines

Journal of Nanobiotechnology volume 22, Article number: 513 (2024) Cite this article

Abstract

The application of nanoscale scaffolds has become a promising strategy in vaccine design, with protein-based nanoparticles offering desirable avenues for the biocompatible and efficient delivery of antigens. Here, we presented a novel endogenous capsid-forming protein, activated-regulated cytoskeleton-associated protein (ARC), which could be engineered through the plug-and-play strategy (SpyCatcher3/SpyTag3) for multivalent display of antigens. Combined with the self-assembly capacity and flexible modularity of ARC, ARC-based vaccines elicited robust immune responses against Mpox or SARS-CoV-2, comparable to those induced by ferritin-based vaccines. Additionally, ARC-based nanoparticles functioned as immunostimulants, efficiently stimulating dendritic cells and facilitating germinal center responses. Even without adjuvants, ARC-based vaccines generated protective immune responses in a lethal challenge model. Hence, this study showed the feasibility of ARC as a novel protein-based nanocarrier for multivalent surface display of pathogenic antigens and demonstrated the potential of exploiting recombinant mammalian retrovirus-like protein as a delivery vehicle for bioactive molecules.

Graphic Abstract

Introduction

Nanoscale scaffolds play a crucial role in antigen delivery to promote the production of efficient and protective immune responses. These scaffolds enable the exhibition of repetitive target antigens, prolong antigen exposure, and enhance the uptake of antigen presentation cells [1,2,3]. Diverse nanoscaffolds, ranging from inorganic material (e.g., gold nanoparticles, silica nanoparticles) to organic material (e.g., polysaccharides, lipid-based nanoparticles, protein-based nanoparticles), have been employed in vaccinology [4,5,6,7,8,9,10]. Among them, self-assembling protein nanoparticles, including virus-like particles (VLPs) and non-virus protein nanoparticles, have been widely utilized as antigen presentation platforms due to excellent biocompatibility and flexible modularity [11, 12].

VLPs have been considered a promising platform in vaccinology to stimulate effective humoral and cellular immune responses, such as approved vaccines against hepatitis B virus (HBV), human papillomavirus (HPV), and hepatitis E virus (HEV) [13,14,15]. Similarly, non-virus self-assembling proteins with organized structures, have been widely used, including ferritin, aquifex aeolicus lumazine synthase, encapsulin, and computational de novo designed proteins (e.g., mI3 and TIP60) [9, 16,17,18,19,20]. Presently, ferritin has been widely employed with various ferritin-based vaccines undergoing tests in clinical trials (NCT05903339, NCT04784767, NCT04645147, NCT03186781) [17]. To further utilize self-assembling protein nanoparticles as universal platforms for antigen display, it is necessary to explore endogenous nanoscaffolds, which could bypass the pre-existing vector immunity or undesired anti-scaffold antibodies [21, 22], for providing alternative options.

It has been reported that endogenous mammalian capsid-forming proteins can self-assemble into virus-like particles to deliver cargoes, showing reduced immunogenicity [23]. Stemmed from a lineage of Ty3/gypsy retrotransposons [24], activated-regulated cytoskeleton-associated protein (ARC) is a highly conserved hub regulator of synaptic plasticity, widely expressed in various species including humans, rats, and drosophila [25]. The structure of ARC is composed of two critical parts, a positively charged N-terminal domain and a negatively charged C-terminal domain, which is homologous to the matrix and capsid domains of the retroviral gag protein [26]. As the unique structure enables gag protein to play a central role in virus assembly [27, 28], ARC has been also demonstrated to self-oligomerize in vitro. However, the potential of ARC as a novel protein-based presentation platform for antigen display is yet to be investigated.

In this study, we envisioned ARC as an endogenous nanoscaffold, capable of surface-display multiple antigens via the plug-and-play display system (SpyCatcher3/SpyTag3) to induce potent humoral and cellular immune responses. Prepared by the Escherichia coli (E. coli) expression system, ARC-based nanoscaffolds significantly enhanced the immunogenicity of conjugated antigens, including receptor binding domain (RBD) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and M1R or A35R of monkeypox (Mpox). Additionally, functioning as immunostimulants, ARC-based vaccines promoted the maturation of dendritic cells, proliferation of B cells, and recruitment of B cells and Tfh cells in the germinal center. Notably, ARC-based vaccines showed favorable safety profiles and low anti-scaffold immunogenicity in mice models. This study provides evidence of ARC to be engineered for diverse antigen delivery, potentially offering an alternative option for vaccine design.

Materials and methods

Experimental cell lines and viruses

Expi293F cells, hACE2-293T stable transfected cells, HeLa cells, and BS-C-1 cells are preserved in our laboratory. Expi293F cells (Thermo Scientific, USA) were cultured in Expi293™ Expression Medium (Thermo Scientific, USA). HeLa cells and hACE2-293T cells were supplemented with 10% fetal bovine serum (FBS, Thermo Scientific, USA), 1% penicillin, and 1% streptomycin in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA). BS-C-1 cells were cultured with 10% fetal bovine serum (FBS, Thermo Scientific, USA), 1% penicillin, and 1% streptomycin in modified Eagle’s medium (Thermo Fisher Scientific). Ectromelia virus strain Moscow (ATCC VR-1374) and Vaccinia virus strain WR (ATCC VR-119 or VACV-EGFP) were propagated in BS-C-1 (ATCC CCL-26) cells.

Preparation of ARC-based nanoparticles and antigenic proteins

The gene of hARC (AAF07185.1) with a GST tag at the N-terminal and a StrepII tag at the C-terminal was chemically synthesized and cloned into the pET 21a + vector. Ferritin (AAI05803.1) with a StrepII tag at the C-terminal was chemically synthesized and cloned into the pET 21a + vector to construct plasmids. Similarly, SpyCatcher3 (SC3: VTTLSGLSGEQGP SGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHT) was fused at the N-terminal of ARC or Ferritin, with a StrepII tag at the C-terminal, and then cloned into the pET 21a + vector. TAT (TAT: GRKKRRQRRRPPQ) was fused at the C-terminal of ARC, with a StrepII tag at the N-terminal, and then cloned into the pET 21a + vector. The constructed plasmids were transformed into E. coli (strain BL21 (DE3)). The transfected E. coli (strain BL21 (DE3)) cell cultures were grown at 37 ℃ with 220 rpm until OD600 reached 0.6–0.8. The temperature was reduced to 16℃, cells were then induced with 1mM isopropyl-b-D-thiogalactopyranoside (IPTG) and grown for 24 h. The cells were collected and lysed by sonicating in 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 1 mM EDTA. The recombinant proteins with Strep II tag in the supernatant of the cell lysates were purified using a 5 ml StrepTrap HP (GE Healthcare, Sweden) chromatography according to the manufacturer’s instructions. Regarding ARC, the protein after being purified by the StrepTrap HP was then incubated with GSTarp 4B (GE Healthcare, Sweden). After cleaving with HRV 3 C protease (SinoBio, China) to remove the GST tag, the protein was purified by Superose 6 10/300 GL column (GE Healthcare, Sweden), and was exchanged into PBS.

The gene of RBD of SARS-CoV-2 (YP_009724390.1, 319–537 aa), M1R of Mpox (QJQ40223.1, 1-181 aa), or A35R of Mpox (QJQ40286.1, 59–181 aa) were chemically synthesized and were cloned into pcDNA3.4. SpyTag3 (ST3: RGVPHIVMVDAYKRYK) was fused at the C-terminal of M1R, RBD, and A35R, with a His tag at the N-terminal, and then cloned into pcDNA3.4. Plasmids were extracted by using Plasmid Maxi Kit (QIAGEN, USA) and transfected into Expi293F cells using Turbofect transfection reagents (Thermo Fisher, USA). The recombinant proteins in the supernatant of the cell lysates were purified using a 5 ml HisTrap HP (GE Healthcare, Sweden) chromatography according to the manufacturer’s instructions. Different molar ratios of RBD-ST3, M1R-ST3, or A35R-ST3 were co-incubated with SC3-ARC overnight at 4℃ to prepare RBD-ARC, M1R-ARC, or A35R-ARC in the PBS buffer, respectively. The forming nanoparticles were then purified using the Superose 6 10/300 GL column (GE Healthcare, Sweden). Purified RBD, M1R, A35R, RBD-ARC, M1R-ARC, and A35R-ARC were then confirmed by SDS-PAGE. The RBD-ferritin, M1R- ferritin, or A35R- ferritin were also prepared as the abovementioned.

Characterization of protein nanoparticles

The particle size and zeta potential of the protein nanoparticles were determined using a Malvern Zetasizer Nano Series equipped with a 4 mW He-Ne ion laser (λ = 633 nm). Besides, the electron microscopy data collection was performed on a Thermo Fisher Talos L120C 120 kV transmission electron microscope, using a CETA direct electron counting detector. Data collection was carried out in the super-resolution mode using the SerialEM software. For cell internalization studies, genetic fusion or tag coupling TAT-ARC nanoparticles were fluorescently labeled with FITC. HeLa cells were seeded in a 96-well plate (Corning, USA) with 2 × 104 cells/per well, culturing in 37℃, 5% CO2 incubator overnight. The labeled nanoparticles were co-cultured with HeLa cells for 8 h to determine cell fluorescence intensity using the Cytation1 imaging reader (Bio Tek, USA).The relative uptake efficiency was calculated by:

$$\text{The}\:\text{uptake}\,\text{efficiency}\:\left(\%\right)\:=\frac{\text{P}ositive\:cells\:of\:FITC}{\text{P}ositive\:cells\:of\:DAPI}*100$$

Surface plasmon resonance assays

The binding of RBD to human ACE2-Fc was measured in the BIAcore 8 K (GE Healthcare, UK) at 25 °C. Briefly, the ACE2-Fc (Sino Biological, China) was immobilized on a protein A chip (Cytiva, USA) to capture the modified RBD for evaluating kinetic parameters (Kon and Koff) and affinities (KD). The KD value of the RBD mutant’s binding affinity for ACE2 was calculated from all the binding curves based on their global fit to a 1:1 binding model using BIA evaluation 4.1 (GE Healthcare, UK).

Animal experiments

Female BALB/c mice (6–8 weeks old) were purchased from Vital River Laboratory Animal Technology (Beijing, China) and lived in specific pathogen-free conditions. All animals were treated following ethical standards and were approved by the Animal Care and Welfare Ethics Committee of Beijing Institute of Biotechnology (ethics number: IACUC-SWGCYJS-2023-007). BALB/c mice were randomized into groups and vaccinated intramuscularly twice with 100 µL/per mouse candidate antigens adjuvanted with or without Alum in the PBS. Mice also received PBS and SC3-ARC as the negative control. Blood was collected from the tail vein on day 14, 28, and 42, and centrifuged at 3000 rpm to obtain serum for subsequent determination of antigen-specific antibody titers. M1R-immunized mice were challenged by intraperitoneal injection of 200 PFU of Ectromelia virus post the boost immunization.

Enzyme-linked immunosorbent assays (ELISA)

RBD, M1R, or A35R (Sino Biological, China) were coated in 96-well plates (Corning, USA) using 50 mM carbonate buffer (pH = 9.6) at a concentration of 1 µg/mL and incubated overnight at 4 °C. After washing with PBST (PBS with 0.1% Tween 20), plates were blocked for 2 h at 37 °C by adding 150 µL of PBS with 2% BSA. Then, the plates were washed three times with PBST. Subsequently, serum was three-fold diluted using dilution buffer (PBS with 0.2% BSA), and incubated at 37 °C for 1 h. Plates were washed three times with PBST and added a 1:20,000 dilution of HRP-conjugated goat anti-mouse IgG (Abcam, UK) at 100 µL/well, incubated for 45 min at 37° C. After washing, TMB solution (Solarbio Life Sciences, China) was added at 100 µL/well. ELISA Stop Solution (Solarbio Life Sciences, China) was then added at 50 µL/well in 10 min. The absorbance was measured at 450 nm/630 nm.

ELISpot evaluation

The Mabtech Ferret IFN-γ ELISpot Kit (Mabtech, Sweden) was used following the manufacturer’s instructions. Briefly, 4 × 105 splenocytes were isolated from immunized mice on day 42 to stimulate in vitro with or without the M1R peptides pool for 48 h at 37 °C, 5% CO2. The plates were washed 5 times with PBS, and biotinylated antibodies were added and incubated for 2 h at room temperature. After washing with PBS 5 times, the HRP secondary antibody was added and incubated at room temperature for 1 h. After washing, TMB chromogenic solution was added for 6–8 min at 37 °C, then washed with plenty of deionized water to stop the chromogenic reaction. Spots of cells expressing IFN-γ were assayed and quantified by an ELISpot reader (SinSage Technology, China).

Multiplex cytokine assays (MCA)

Isolated splenocytes were added into 96-well cell culture plates and incubated with or without the M1R peptides pool for 2 days at 37 °C, 5% CO2. Secreted cytokines in the culture medium were analyzed by Luminex-based Bio-Plex Pro Mouse Cytokine 23-plex Assay (Biorad, USA) according to the manufacturer’s introductions.

Chemiluminescence immunoassays

The magnetic particles coated with RBD bound to the neutralization antibodies in the sample to form immune complexes on the surface of the magnetic beads. The ACE2 protein labeled with acridinium ester (AE), a chemiluminescent agent, competed with the antibodies of the blood sample during experiments. After the incubation, magnetic beads were precipitated to detect chemiluminescent signals. Briefly, serially diluted SARS-CoV-2 neutralizing antibodies (Vazyme, China) (20, 6.67, 2.22, 0.74, 0.25, 0.08, 0.03, 0 µg/mL) were used to plot the standard curve. The serum from immunized mice was twenty-fold diluted for further determination. All samples were detected to quantify their ability to block the interaction between RBD and hACE2 using the automatic chemiluminescence analyzer (RealMind, China).

SARS-CoV-2 Pseudovirus neutralization assays

50 µL serum was inactivated at 56 °C for 30 min and three-fold diluted in 96-well plates (Corning, USA). 50 µL of WT pseudovirus was added and incubated for 1 h at 37 °C, 5% CO2. Next, 100 µL ACE2-293T cells (2 × 105 cells/mL) were added and incubated for 48 h. The intensity of luciferase luminescence was measured after lysing the cells. The neutralization titer (NT50 titer) was defined as the endpoint dilution reaching 50% of the luminescence readings of virus-only control wells.

Live ECTV or VACV neutralization assays

Serially diluted serum was incubated with Ectromelia virus (ECTV) or VACV-EGFP at 37 °C for 1 h. The serum-virus complexes were added into BS-C-1 cell monolayers cultivated in 96-well plates and subjected to a further 48 h of incubation. Cells treated with ECTV were stained with 0.05% crystal violet for 40 min. Optical density (OD) was measured at 570 nm/630 nm after adding the decolorization solution. Cells treated with VACV-EGFP were stained with Hoechst 33,258 (Thermo Fisher, USA). Cellular fluorescence intensity of EGFP was measured by an imaging reader (Cytation 1, USA). The neutralization titers were defined as the dilution of the serum that reached 50% of the OD570 (ECTV) or fluorescence (VACV-EGFP) of controls with no virus.

In vitro BMDC activation assays

Bone marrow cells were collected from the tibias of 10-week BALB/c mice. Filtered by 70 μm meshes, cells were treated with red blood cell lysis buffer (Sigma-Adlrich, USA) according to the manufacturer’s introductions. Viable cells were adjusted to 2 × 107 cells/mL, cultured in RPMI-1640 (Gibco, USA) supplemented with 20 ng/mL GM-CSF (Proteintech, USA) and 10 ng/mL IL-4 (Proteintech, USA) at 37 °C, 5% CO2. One-half of the medium was replaced every 2 days. On the sixth day, BMDCs were collected and resuspended in a complete RPMI-1640 medium.

BMDCs were cultured with equimolar quantities of M1R (10 µg/mL, 2mL) in the form of M1R-ST3 or M1R-ARC in 6-well microplates (Corning, USA). 0.1 µg/mL LPS (Solarbio Life Science, China) and PBS were added as the positive control or the negative control, respectively. After incubating for 20 h, the cells and supernatant were collected. BMDCs were stained with fluorescence antibodies purchased from BioLegend, Inc. (CA, USA), including BV605 anti-mouse CD11c antibody, PE-CY7 anti-mouse CD80 antibody, BV421 anti-mouse CD86, PE anti-mouse CD40 antibody, and AF647 anti-mouse MHCII antibody, and the viability dye Zombie NIR to exclude dead cells from data analysis. The data were collected on a FACS CantoTM (BD Biosciences, USA) and analyzed using FlowJo software (version 10.8.1). In addition, the supernatant was determined by TNF-α, IL-6, and IL-1β ELISA kits (Absin, China) based on the manufacturer’s protocols.

In vivo DC maturation assays

36 h after intramuscular immunization, single-cell suspensions isolated from LNs were prepared. DC maturation was assayed by determining the expression of CD40, CD80, CD86, MHCII, or MHCI levels via FACS CantoTM (BD Biosciences, USA) following the BMDC gating strategy.

Germinal center response assays

Spleens were harvested 7 days post-boost immunization and treated with red blood cell lysis buffer (Sigma-Adlrich, USA) according to the manufacturer’s introductions. 2 × 106 splenocytes were stained with a mixture of antibodies against lineage markers, including BV605 anti-mouse PD-1 antibody, PE anti-mouse CXCR5 antibody, FITC anti-mouse CD4 antibody, PerCP/Cy5.5 anti-mouse B220 antibody, APC anti-mouse GL7 antibody, BV421 anti-mouse Fas antibody, and AF700 anti-mouse CD3 antibody, and the viability dye Zombie NIR to exclude dead cells from data analysis. The cells were assayed by FACS CantoTM (BD Biosciences, USA) and data were analyzed using FlowJo software (version 10.8.1).

In vivo imaging

BALB/c mice were administered a single dose of Alexa Fluor 750-labeled antigens adjuvanted with or without Alum. In vivo imaging was performed at different time points (4, 8, 24, 48, 72 h) using the PerkinElmer IVIS system. The fluorescence intensity of Alexa Fluor 750-antigens in inguinal lymph nodes at 48 h was also conducted. Fluorescence signals in the regions of interest were analyzed using ART OPTIX-OptiView software (version 2.02.01).

Single-cell sequencing analysis

Single cells isolated from LNs or spleens were paired with Gel Beads-in-emulsion (GEMs) using the 10x Chromium 3′ v3 chemistry system (10x Genomics) according to the manufacturer’s introductions. The sequencing and bioinformatic analysis were performed on the platform of Majorbio Co., Ltd (Shanghai, China).

Safety assessments

Mice were vaccinated with equimolar quantities of M1R (5 µg) in the form of M1R-ST3 or M1R-ARC, adjuvanted with Alum (50 µg) at 2-week intervals (day 0 and 14), with PBS as the negative control. Following 7 days after the second immunization, mice were euthanized for the retrieval of serum, kidney, and liver samples. Serum samples were subjected to a comprehensive analysis of blood chemistry parameters, including alanine aminotransferase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN), and alkaline phosphatase (ALP), creatine (CR), total Bilirubin (TBil), total globulin (TG), and total protein (TP). Organ specimens were collected for histological examination.

Statistical analysis

Statistical analysis was performed with the GraphPad Prism 9.0.0 software. The antibody titers were presented in a log10-transformed format for analysis. Antibody titers, cytokine levels, radiant intensity, and blood biochemistry were compared between groups or time points using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test or unpaired t-test. Survival curves were determined via simple survival analysis (Kaplan-Meier). p < 0.05 was considered significant (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant).

Results

Preparation and characterization of the ARC-based protein nanoparticles

Full-length human ARC was produced in the Escherichia coli BL21 (DE3) strain. The expected molecular weight, approximately 55 kDa, was validated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot (WB) (Fig. 1A). Dynamic lighter scatter (DLS) assays showed that the diameter of the ARC nanoparticle was 22.78 ± 1.93 nm (Fig. 1C and E) with polydispersity index 0.26 (Fig. S1A), which was verified by transmission electron microscope (TEM) (Fig. 1B). Furthermore, SDS treatment significantly disrupts ARC nanoparticles and ferritin nanoparticles into smaller sizes (Fig. 1C), indicating the self-assembly of ARC was achieved through similar intermolecular interactions as observed in ferritin.

Fig. 1
figure 1

The characterization of ARC-based nanoparticles. (A) The SDS-PAGE and western blot (WB) of ARC. (B) The transmission electron microscope images of ARC. Scale bar, 100 nm. (C) The size of BSA, Ferritin, and ARC before or after 0.5% SDS treatment, n = 5. (D) Schematic illustration of vaccine design based on ARC nanoparticles. (E) The size distribution of ARC and SC3-ARC. (F) The SDS-PAGE of TAT-ARC. (G) The zeta potential of SC3-ARC, and TAT-ARC, n = 3. (H) The images of cell internalization of TAT-ARC. Scale bar, 200 μm. Data are the mean ± standard error of the mean (SEM). The unpaired t-test in (C). ***p < 0.001, ****p < 0.0001; not significant, p>0.05

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The modification of protein nanoparticles for antigen presentation can be accomplished through genetic fusion or tag coupling (e.g. the SpyCatcher3/SpyTag3 (SC3/ST3) system) [29,30,31,32,33,34,35]. To initially investigate the presentation capacity of ARC, we used TAT peptides derived from HIV-1 to conjugate with ARC (Fig. 1D). We prepared SC3-ARC with the expected molecular mass of ~ 70 kDa (Fig. S1B). The increased size of SC3-ARC relative to ARC (Fig. 1E), as characterized by DLS, was consistent with TEM (Fig. S1C), and the polydispersity index is ~ 0.24 (Fig. S1A). To examine the stability of SC3-ARC scaffolds, nanoparticles were frozen at -80 °C for 10 min, thawed in water at room temperature repeating 1 or 3 times, then detected their size distributions. The results indicated that SC3-ARC maintained similar size distributions in intensity with no unwanted aggregations (Fig. S1D). Additionally, the polydispersity index data (Fig. S1E) and size distributions of ARC nanoparticles (Fig. S1F) under 4℃ have no significant difference comparing day 0 or six months.

The TAT peptides are known for their highly positive charges and belong to the category of cell-penetrating peptides [36, 37]. As evident from SDS-PAGE and zeta potential (Fig. 1F and G), a clear molecular shift and a slight increase of zeta potential of TAT-ARC were observed, indicating the successful loading of TAT peptides on ARC nanoparticles. Furthermore, TAT assisted in the internalization of FITC-labeled ARC into cells (Fig. 1H), with the SC3/ST3 system-based TAT-ARC exhibiting a stronger signal intensity than that of genetic fusion-based TAT-ARC (Fig. S1G), thereby suggesting that tag coupling was optimal for engineering ARC nanoparticles to present candidate antigens.

ARC-based nanoparticles enhance the immunogenicity of RBD of SARS-CoV-2

To evaluate the potential of ARC as nanoscaffolds for presenting recombinant proteins, the receptor binding domain (RBD) of SARS-CoV-2 was exhibited on ARC using the SC3/ST3 system. RBD-ARC was prepared at the optimal molar ratio of RBD-ST3:SC3-ARC = 2:1 (Fig. S1H). After purification using size exclusion chromatograms (Fig. S1I), we then characterized the conformational exhibition of modified RBD by examining binding kinetic and affinity with the human angiotensin-converting enzyme 2 (hACE2) receptor. Surface plasmon resonance assays indicated that RBD-ST3 had a similar dose-dependent manner with the hACE2 receptor compared to RBD, showing ST3 didn’t interfere with the conformation of RBD (Fig. 2A, B and D). Notably, the binding affinity of RBD-ARC was significantly higher than that of RBD, indicating that ARC nanoparticles could multivalently display antigens with proper exposure (Fig. 2C and D).

Fig. 2
figure 2

ARC-based nanoparticles enhance the immunogenicity of RBD of SARS-CoV-2. (A-C) Surface plasmon resonance (SPR) assays of RBD, RBD-ST3, and RBD-ARC. (D) The association constant (ka), dissociation constant (kd), and affinity constant (KD) of RBD, RBD-ST3, and RBD-ARC with hACE receptor. (E) Schematic illustration of mice vaccination and blood collection. BALB/c mice were immunized with equimolar quantities of RBD (5 µg) in the form of RBD-ST3 or RBD-ARC, all adjuvanted with Alum (50 µg) at 2-week intervals (day 0 and 14). Control mice were received with PBS or SC3-ARC. (F) RBD-specific IgG titers measured at day 14, 28, and 42, n = 6. (G) RBD-specific IgG1 and IgG2a titers measured at day 28, n = 6. (H) The concentration of SARS-CoV-2 neutralizing antibodies determined by chemiluminescence immunoassay at day 28, n = 6. (I) Neutralization titers at 50% inhibition (NT50) against SARS-CoV-2 pseudoviruses measured at day 28. n = 6. Data are the mean ± standard error of the mean (SEM). Data shown in F and G were determined via two-way ANOVA with multiple comparisons. Data shown in H and I were determined via one-way ANOVA with Tukey’s multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

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Subsequently, BALB/c mice were intramuscularly (i.m.) immunized with two doses of RBD, RBD-ST3, and RBD-ARC, all adjuvanted with aluminum (Alum), at 2-week intervals (Fig. 2E). Immunized serums were collected to examine RBD-specific IgG titers at day 14, 28, and 42, respectively. We initially showed that RBD-ST3 did not affect the immunogenicity of RBD (Fig. S2A-S2C). RBD-ARC induced significantly higher specific IgG titers compared to RBD-ST3 at different time points, with a 16.2-fold increase at day 28 (Fig. 2F). In contrast to RBD-ST3, RBD-ARC induced substantially increased levels of IgG2a (Fig. 2G), the ratios of IgG1/IgG2a more balanced.

To further assess the immunogenicity of ARC-based vaccines, a chemiluminescence assay based on blocking the interaction between RBD and hACE2 was used to measure the concentration of neutralizing antibodies (Nab) in serum, revealing RBD-ARC induced a 5.6-fold increase of Nab relative to RBD at 14 days post-boost immunization (Fig. 2H). Moreover, pseudovirus neutralization assays also showed that RBD-ARC induced higher neutralization titers against SARS-CoV-2 compared to RBD-ST3 (Fig. 2I). After the second dose, RBD-ARC exhibited a geometric mean neutralization titer of 1230, 56-fold higher than that of RBD-ST3.

ARC-based nanoparticles enhance the immunogenicity of M1R of Mpox

To assess the universal applicability of ARC-based nanoparticles for vaccine development, M1R derived from the Monkeypox virus was used as another model antigen. M1R-ARC was prepared at the optimal molar ratio of M1R-ST3:SC3-ARC = 1:1 (Fig. S1J). The characterization of M1R-ARC by DLS showed that M1R-ARC still exhibited nanosized conformation (Fig. S1K). Mice were intramuscularly (i.m.) immunized with two doses of M1R, M1R-ST3, and M1R-ARC, all adjuvanted with Alum, at 2-week intervals (Fig. 3A). Similar to what was observed in RBD-ST3, the ST3 fusion also did not affect the immunogenicity of M1R (Fig. S2D-S2F). Compared to M1R-ST3, M1R-ARC elicited more robust M1R-specific immune responses at different time points, with a 58.7-fold increase at day 28 (Fig. 3B). Additionally, even at one-tenth of the dose of M1R-ST3, M1R-ARC also induced higher M1R-specific IgG titers (Fig. 3B), demonstrating the ability of ARC-based nanoparticles to stimulate strong immune response with a lower antigen dosage.

Fig. 3
figure 3

ARC-based nanoparticles enhance the immunogenicity of M1R of Mpox. BALB/c mice were immunized with equimolar quantities of M1R (5 µg) in the form of M1R-ST3 or M1R-ARC, all adjuvanted with Alum (50 µg) at 2-week intervals (day 0 and 14). Control mice were received with PBS or SC3-ARC. (A) Schematic diagram of mice vaccination, sample collection, and challenge. (B) M1R-specific IgG titers of mice, immunized with M1R-ARC in a M1R-dose dependent manner, measured at day 14, 28, and 42, n = 6. (C) Serum neutralization NT50 titers against Ectromelia virus (ECTV) assessed at day 28, n = 6. (D) M1R-specific IgG subtype titers measured at 28 d, n = 6. (E) Cytokines secreted by stimulated splenocytes of immunized mice measured at day 21, n = 5. (F) IFN-γ-secreting splenocytes of immunized mice measured at day 21, n = 5. (G-H) Germinal center responses in spleens of immunized mice at day 21, n = 5. (I) Survival curves of immunized mice challenged by ECTV at day 42, n = 6. Data are the mean ± standard error of the mean (SEM). Data shown in C, G, and H were determined via one-way ANOVA with Tukey’s multiple comparisons. Data shown in B was determined via two-way ANOVA with multiple comparisons. Data shown in D was determined via the unpaired t-test. Data shown in I were determined via simple survival analysis (Kaplan-Meier). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; not significant, p>0.05

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We next applied Extromelia virus (ECTV) to measure the neutralizing antibody titers induced by vaccination, owing to ECTV closely resembling the genetic and disease characteristics of the monkeypox virus [38]. M1R-ARC elicited higher neutralizing antibody titers than M1R-ST3, with a 3.8-fold increase at day 28 (Fig. 3C). In line with the RBD-ARC vaccine, M1R-ARC also stimulated a Th1-skewing humoral immune response (Fig. 3D), which was further verified by multiplex cytokine assays, showing significantly higher levels of GM-CSF, IFN-γ, IL-2, and TNF-α compared to M1R-ST3 (Fig. 3E). Moreover, the quantification of T-cell responses was achieved through enzyme-linked immunospot (ELISpot). M1R-ARC vaccination significantly increased the expression of IFN-γ relative to M1R-ST3, inducing approximately 150 spots per million splenocytes (Fig. 3F).

To further explore the intrinsic mechanism of potent immune responses elicited by ARC-based nanoparticles, we performed flow cytometry to assess germinal center (GC) responses at day 7 post-2nd immunization. GC B cells or T follicular helper (Tfh) cells were sorted by GL7 + Fas + in the B cells population or CXCR5 + PD-1 + in the CD4 + T cells population, respectively. The enhanced humoral response induced by M1R-ARC was evidenced by a notable rise in the proportion of GC B cells (Fig. 3G) and a moderate increase in the proportion of Tfh cells (Fig. 3H). Notably, in the lethal challenge model, M1R-ARC provided 68% protection efficiency against ECTV. In contrast, all mice administered with PBS or M1R-ST3 died within 7 days or 18 days, respectively (Fig. 3I).

ARC nanoparticles efficiently stimulate DC maturation and prolong antigen retention in vivo

Dendritic cells (DCs) play vital roles in efficient processing and cross-presentation of specific antigens. Specific antigens conjugated with protein nanoparticles pronouncedly enhance antigen uptake by DCs owing to the larger size of nanoparticles [39,40,41]. To explore whether ARC-based nanoparticles could activate DCs like other protein nanoparticles in vitro, M1R-ST3 or M1R-ARC were added into BMDCs and incubated for 24 h. PBS and lipopolysaccharide (LPS) were used as the negative and positive control, respectively. The Limulus Amebocyte lysate (LAL) endotoxin assays demonstrated that all proteins have negligible endotoxin contamination (Fig. S3A). Cells were harvested for flow cytometry analysis, and the supernatants were collected for cytokines determination. M1R-ARC strikingly activated maturation of BMDCs with high-level expressions of CD40, CD80, and CD86 (Fig. 4A-C and Fig. S3B-D), as well as increased proinflammatory cytokines in supernatants, including TNF-α, IL-6, and IL-1β (Fig. 4D-F).

Fig. 4
figure 4

ARC-based nanoparticles efficiently stimulate the maturation of DCs in vitro and in vivo. (A-C) BMDCs were treated with equimolar quantities of M1R (10 µg/mL, 2 mL) in the form of M1R-ST3 or M1R-ARC for 20 h. PBS and lipopolysaccharide (LPS) were used as the negative and positive control, respectively. CD86, CD80, and CD40 expressions were analyzed by flow cytometry, n = 3. (D-F) The concentration of TNF-α, IL-6, and IL-1β secreted by BMDCs in the culture medium, n = 3. (G-I) BALB/c mice were immunized with equimolar quantities of M1R (5 µg) in the form of M1R-ST3 or M1R-ARC, all adjuvanted with Alum (50 µg) at 2-week intervals (day 0 and 14). Control mice were received with PBS or SC3-ARC. The proportions of MHCI + CD80+, MHCII + CD80+, and CD80 + CD86 + cells in DCs of inguinal lymph nodes were measured at 36 h post-boost immunization, n = 5. (J) Fluorescence intensity of Alexa Fluor 750-antigens in inguinal lymph nodes at 48 h, n = 3. (K) In vivo imaging of Alexa Fluor 750-antigens in mice at different time points (0, 8, 24, 48, 72 h), n = 3. (L) Normalized total radiant efficiency of Alexa Fluor 750-antigen at different time points (0, 8, 24, 48, 72 h), n = 3. Data are the mean ± standard error of the mean (SEM). Data shown in D-I were determined via one-way ANOVA with Tukey’s multiple comparisons. Data shown in L were determined via two-way ANOVA with multiple comparisons. *p < 0.05, **p < 0.01, ****p < 0.0001; not significant, p>0.05

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The activation of specific immune responses in vivo relies on the promotion of antigen uptake by dendritic cells and their migration into lymph nodes (LNs) [42]. To further demonstrate the effectiveness of ARC-based nanoscaffolds in stimulating DC activation in LNs, we collected inguinal lymph nodes to analyze DC maturation 36 h after the 2nd immunization with PBS, M1R-ST3, or M1R-ARC. Flow cytometry was performed to detect the expressions of costimulatory molecules and MHC molecules in the CD11c + cell population. M1R-ARC significantly increased co-expressions of MHCI + CD80+, MHCII + CD80+, or CD80 + CD86 + in DCs compared to M1R-ST3 (Fig. 4G-I). We next evaluated local depot effects through intramuscular vaccination with Alexa Fluor 750 labeled M1R-ST3 and M1R-ARC. The tissue distribution of fluorescence mainly accumulated in the liver, kidney, and draining lymph nodes (dLNs) (Fig. S3E), of which a stronger fluorescence of M1R-ARC in proximal inguinal LNs was enriched (Fig. 4J). The fluorescence of M1R-ST3 and M1R-ARC in livers and kidneys decreased to a similar intensity at 72 h. (Fig. S3E). Meanwhile, in vivo imaging results revealed that M1R-ST3 was rapidly cleared, with only 20% remaining at the injection sites after 8 h, whereas M1R-ARC exhibited sustainable release at different time points (8, 24, 48, and 72 h) (Fig. 4K and L), indicating that ARC-based nanoscaffolds could enhance antigen retention time at injection sites, prolonging antigen accumulation and promoting DCs maturation in vivo.

Single-cell transcriptomics of ARC-based vaccines in innate and adaptive immunity

Following the observations of the activation of DCs in dLNs and the enhancement of GC response in spleens after vaccination with ARC-based nanoparticles, we proceeded to characterize the transcriptional landscape of innate and adaptive immunity (Fig. 5A). To investigate the cellular and molecular mechanisms involved in the enhanced immunogenicity of ARC-based nanoparticles, we isolated approximately 10,000 single cells from the dLNs at 36 h post-1st vaccination with PBS, M1R-ST3, or M1R-ARC. B cells, T cells, DCs, macrophages, and NK cell populations were defined upon clustering and tSNE embedding (Fig. 5B). A notable increase in the number of differentially expressed genes (DEGs) of DC and macrophage clusters was detected in M1R-ARC compared to M1R-ST3. In selected genes, M1R-ARC substantially upregulated the genes involved in defense response to viruses (Ifngr1, Cd86, Cd226, Stat5b), antigen processing and presentation (Marchf1, H2-oa, H2-ob, Tapbp), and cell differentiation (Bcl11b, Gpr183) over that of M1R-ST3 and PBS (Fig. 5C). Gene Ontology (GO) analysis further demonstrated that M1R-ARC positively regulated immune responses related to antigen processing and presentation (Fig. 5D), confirming the enhancement of antigen presentation cell processing for eliciting potent immune responses.

Fig. 5
figure 5

Single-cell transcriptomics of ARC-based vaccines in innate immunity and adaptive immunity. BALB/c mice were immunized with equimolar quantities of M1R (5 µg) in the form of M1R-ST3 or M1R-ARC, all adjuvanted with Alum (50 µg). Control mice were received with PBS or SC3-ARC. (A) Schematic diagram of single-cell analysis. (B) Annotation of cell types clustered by single-cell transcriptional analysis in inguinal lymph nodes at 36 h post prime immunization. (C) Heat map of key expressed genes in DCs and macrophages in inguinal lymph nodes at 36 h post prime immunization. (D) Gene ontology analysis of upregulation genes expression of DCs and macrophages in inguinal lymph nodes at 36 h post prime immunization. (E) Heat map of key expressed genes in B cells at 14 days post boost immunization. (F) Gene ontology analysis of upregulation gene expression of B cells at 14 days post boost immunization

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Additionally, we also conducted a transcriptomic analysis of spleens at 7 days post-2nd immunization, isolating 10,000 single cells and clustering them into 33 cell subsets. In contrast to the native immunity of dLNs at 36 h post-priming, the DEGs of adaptive immunity were reduced. However, the genes involved in antigen processing via MHC I (H2-K1, H2-T23), response to cytokines (Cd2, Ifinar1), B and T cell activation and proliferation (Mzb1, Cd81, Ccr7, Satb1) were more highly expressed in the M1R-ARC group compared to the M1R or control group (Fig. 5E). Moreover, GO analysis verified that ARC-based nanoparticles induced mature B cell differentiation and positive regulation of T cell proliferation, stimulating antibodies isotype switching and the production of more specific IgG compared to M1R-ST3 (Fig. 5F).

ARC-based vaccines provide comparable immune responses relative to ferritin-based vaccines

Ferritin, composed of 24 subunits that self-assemble into a hollow, spherical protein cage, has been widely applied in recombinant antigen design to enhance immune responses [43,44,45,46]. In this study, the immunogenicity of ARC-based vaccines was compared with that induced by ferritin-based vaccines. Analysis of IgG titers elicited by different antigens (including RBD, M1R, and A35R) in BALB/c mice revealed that antigen-specific antibodies induced by ARC-based vaccines obtained no statistical difference relative to ferritin-based vaccines post-2nd vaccination (Fig. S4A-S4C). Similarly, both M1R-ARC and M1R-Ferritin elicited a more balanced Th1/Th2 immune response (Fig. S4D), with comparable performance in provoking cytokines production (Fig. S4E). Furthermore, for the neutralizing antibodies against SARS-CoV-2 (Fig. S4F) or ECTV (Fig. S4G), as well as the in vivo protective efficacy against the lethal challenge of ECTV (Fig. S4H), ARC-based vaccines and ferritin-based vaccines did not exhibit statistical significance. Additionally, to measure the capacity of co-display multiple antigens, we selected M1R and A35R, co-conjugated with ARC or ferritin, and compared their immunogenicity. When compared to M1R-ST3 or A35R-ST3, both ARC- and ferritin-based divalent vaccines induced higher levels of antigen-specific antibodies (Fig. S5A, S5B) and neutralization titers against Vaccinia virus (VACV) (Fig. S5C).

Due to the ability of ARC nanoparticles to activate DCs in vitro, we further explored their adjuvant activity in vivo. Fluorescence imaging revealed that the antigen retention time of M1R-ARC was comparable, whether it was administered alone or in combination with Alum (Fig. 6A and B). While the addition of Alum increased the immunogenicity of M1R, its presence or absence did not have a significant impact on the antibody response induced by M1R-ARC (Fig. 6C). Notably, the adjuvant-free M1R-ARC elicited higher M1R-specific IgG titers (Fig. 6D) and higher levels of neutralizing antibodies against VACV (Fig. 6E, Fig. S6A) compared to M1R-Ferritin. Additionally, the RBD-specific IgG titers and neutralizing antibodies against SARS-CoV-2 elicited by RBD-ARC were similar to those induced by the ferritin-based vaccines (Fig. S7A, S7B), and both were significantly higher than RBD-ST3. Importantly, compared to the M1R-ST3 group, where all mice succumbed within 10 days, the M1R-ARC vaccination provided complete protection against the lethal challenge of ECTV (Fig. 6F). Furthermore, ARC-based vaccines induced a more balanced Th1/Th2-biased humoral response compared to ferritin-based vaccines (Fig. 6G and Fig. S7C), with both stimulating a high-level T cell response without the presence of Alum (Fig. 6H).

Fig. 6
figure 6

ARC-based vaccines efficiently stimulate immune responses adjuvanted without adjuvants. BALB/c mice were immunized with equimolar quantities of M1R (5 µg) in the form of M1R-ST3, M1R-ARC, or M1R-Ferritin, adjuvanted with (C) or without (C-H) Alum (50 µg) at 2-week intervals (day 0 and 14). Control mice were received with PBS. (A) In vivo imaging of Alexa Fluor 750-antigens at different time points after immunization, n = 3. (B) Normalized fluorescence intensity of Alexa Fluor 750-antigens at different time points (0, 8, 24, 48, 72 h), n = 3. (C) M1R-specific IgG titers of mice immunized with M1R-ST3 or M1R-ARC formulated with or without Alum measured at day 14, 28, and 42, n = 6. (D) M1R-specific IgG titers of mice immunized with M1R-ST3, M1R-ARC, or M1R-Ferritin adjuvanted without Alum measured at day 14, 28, 42, n = 6. (E) Serum neutralization titers against Vaccinia virus expressing EGFP assessed at day 28, n = 6. (F) Survival curves of immunized mice challenged by ECTV at 30 days after boost immunization, n = 6. (G) M1R-specific IgG1/IgG2a ratios measured at day 28, n = 6. (H) IFN-γ-secreting splenocytes of immunized mice measured at 14 days after boost immunization, n = 5. Data are the mean ± standard error of the mean (SEM). C and D were determined via two-way ANOVA with multiple comparisons. E, G, and H were determined via one-way ANOVA with Tukey’s multiple comparisons. Data shown in G were determined via simple survival analysis (Kaplan-Meier). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; not significant, p>0.05

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Safety evaluation of ARC-based vaccines

To assess the safety profile of ARC-based vaccines, BALB/c mice were vaccinated with either M1R-ST3 or M1R-ARC, both adjuvanted with Alum. Blood biochemistry and histopathological examination of kidneys and livers were analyzed on day 7 after boost immunization. The biochemical markers such as aspartate glutamate transaminase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), blood urea nitrogen (urea), creatine (CR), total bilirubin (TBil), total globulin (TG), and total protein (TP) in mice immunized with M1R-ARC showed no significant differences compared to the control group (Fig. 7A). Moreover, the absence of vascular congestion and inflammatory cell infiltration suggested that there were no significant histopathological alterations in the liver and kidney tissues (Fig. 7B). Of note, purified human ARC nanoparticles exhibit low-level production of anti-ARC antibodies (Fig. 7C) without influencing M1R-specific antibody titers, which the extent of anti-scaffold antibody titers was similar to ferritin-based vaccines induced (Fig. S7D), suggesting the feasibility for antigen delivery in mice models.

Fig. 7
figure 7

The safety profile of ARC-based presentation platform. BALB/c mice were immunized with equimolar quantities of M1R (5 µg) in the form of M1R-ST3, or M1R-ARC, all adjuvanted with Alum (50 µg) at 2-week intervals (day 0 and 14). Control mice were received with PBS. (A) Analysis of blood biochemistry at 7 days post boost immunization, n = 5. (B) The representative hematoxylin and eosin (H&E) images of kidney and liver sections at 7 days post-boost immunization. (C) The anti-ARC IgG titers were measured at 14 days post-2nd immunization, n = 5. Data are the mean ± standard error of the mean (SEM). Data shown in A were determined via one-way ANOVA with Tukey’s multiple comparisons. C was determined via two-way ANOVA with multiple comparisons. *p < 0.05; not significant, p>0.05

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Discussion

Attempts over the years to enhance the immunogenicity of subunit vaccines have led to the advancement of antigen presentation platforms. The display of multivalent antigens based on self-assembling protein nanoparticles enhances the immune system’s recognition by presenting antigens in a virus-like conformation, facilitating their uptake by antigen-presenting cells [9]. The larger antigen size of protein nanoparticles enhances immune responses by increasing complement binding and retention in lymph nodes, resulting in more effective B-cell receptor crosslinking and improved vaccine efficacy [47,48,49]. Consequently, multiple self-assembling protein nanoparticles have been applied in vaccine designs, with ongoing research focused on expanding the range of candidates to offer a wider array of options. This study demonstrated that ARC, an endogenous protein, acts as a novel protein scaffold upon the plug-and-play display of antigens to provide robust and efficient immune responses.

ARC-based nanoparticles were prepared via the E. coli expression system with a size of 22.78 ± 1.93 nm, demonstrating the in vitro self-assembly. ARC is homologous with retroviral Gag proteins and capable of spontaneously forming virus-like structures [24,25,26,27,28]. However, there is still a lack of understanding of the detailed molecular basis for their assembly. Based on the predicted structure of ARC, it has two folded regions, the C-terminal domain (CTD) and the N-terminal domain (NTD) combined with a central linker, and flexible unfolded regions are flanking on both sides. The CTD of ARC includes two homologous domains, probably forming a bilobar structure, N- and C-lobe [24, 50]. They showed the structural homology of the HIV Gag protein capsid domain (CA) [26, 51].ARC also exhibits similar properties to HIV in capsid forming, which is sensitive to salt and phosphate levels [52]. Additionally, the NTD of ARC might evolve from the matrix domain (MA) of Gag polyprotein, mediating binding to membranes, which is similar to retroviral MA [27, 28]. The NTD of ARC contains two positively charged antiparallel helical coils, which have been proven to be necessary and sufficient in the oligomerization of ARC, of which a 7-residue oligomerization motif of NTD, is critical for the capsid forming [27]. Researchers have indicated that the NTD domain supports self-association into the dimer stage and then assembles into larger polymers [51, 53]. We attempted to crystalize ARC for the resolution of the detailed structure using X-ray diffraction but encountered obstacles in ARC crystallization. It might result from the flexible disordered region of ARC. However, Maria et al. speculated that approximately 130 copies of ARC protein may be present per capsid based on the layer density of these capsids [26], which might provide more evidence of ARC self-assembly.

Although in this unclear structural basis context, we firstly applied the SpyCatcher3/SpyTag3 system for surface display of cell-penetrating peptides and heterologous antigens, showing that the plug-and-play strategy was suitable for engineering ARC nanoparticles, which multivalently displayed candidate antigens correctly. ARC-based vaccines demonstrated protective immunity against both SARS-CoV-2 and Mpox. Additionally, ARC nanoscaffolds co-displayed two different antigens, M1R and A35R, which induced stronger immune responses than M1R or A35R alone, again highlighting the feasibility of ARC as a novel and generic protein-based nanoscaffold.

The optimal size for vaccine particles to facilitate efficient trafficking into lymphoid tissues typically ranges from 15 to 100 nm [42, 54,55,56]. These vaccines significantly increased antigen exposure and improved antigen accumulation in vivo, leading to the maturation of DCs, activation of B cells, and enhancement of GC response. Moreover, single-cell sequencing revealed that ARC-based vaccines upregulated genes involved in antiviral defense, antigen processing and presentation, and cell differentiation. Specifically, even in the absence of adjuvant, ARC-based vaccines have been shown to induce potent antigen-specific antibodies with a balanced Th1/Th2-skewing humoral response, which indicated the potential of ARC as immunostimulants for enhancing the immunogenicity of protective antigens.

Virus-like particles (VLPs) are also promising self-assembling protein-based nanoparticle platforms in antigen presentation. Some studies have investigated the capability of Hepatitis B core virus-like particles [57, 58] and Norovirus-derived nanoparticles [56] to display heterologous antigens in their repetitive viral protein surface. They stimulated effective humoral and cellular immune responses against immunogens, yet pre-existing or excessive anti-scaffold antibodies probably constrain their efficacy. ARC, as a novel endogenous capsid-forming protein, combines the feature of high antigen presentation capacity of VLPs and lower immunogenicity of endogenous proteins, which underscored the potential application in biomolecule delivery. Given ferritin, composed of the self-assembling 24-mer subunits, is a classic and endogenous platform to present antigens, which has been widely applied [43,44,45,46]. To preliminarily assess the immunogenicity of ARC-based vaccines, we performed immunological assessments to compare their immune response with ferritin-based vaccines by using the same antigens, antigen doses, vaccination strategies, and mice models. Collectively, the IgG titers and protection efficiency obtained by ARC-based scaffolds were comparable to that induced by ferritin-based. Moreover, without adjuvants, M1R-ARC exhibited stronger immune responses than M1R-Ferritin, potentially attributed to the larger size of ARC relative to ferritin.

ARC showed a good safety profile based on the results of blood biochemistry indexes and negligible vascular congestion and inflammatory cell infiltration of the tissue section. The lower titers of anti-ARC-specific antibodies did not interfere with M1R- or RBD-specific immune responses. Besides, ferritin-based vaccines also induced ferritin-specific antibodies, the titers of M1R- or RBD-specific antibodies were not significantly influenced. Combining the universal application of human ferritin in drug delivery and antigen presentation [59,60,61,62], we supposed the excellent biocompatibility of ARC as a nanoscaffold for antigen delivery.

Conclusion

In this study, our results revealed a new endogenous self-assembling protein, ARC, could be engineered through the plug-and-play strategy (SpyCatcher3/SpyTag3), which multivalently displays antigens with correct conformation and elicits robust and effective immune responses. Even without adjuvants, ARC-based vaccines efficiently stimulate strong immune responses and provide protective immunity in a lethal challenge model. Therefore, these findings indicate that the ARC-based nanoparticles have favorable characteristics for antigen display, providing an alternative option to develop a biocompatible and effective immunogenic subunit vaccine.

Data availability

Data is provided within the manuscript or supplementary information files.

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Acknowledgements

The authors thank D. Wang, C. F. Mao, and F. X. Zhao for their valuable comments and suggestions.

Funding

This work was supported in part by Beijing Nova Program of Science and Technology (20220484127) and National Natural Science Foundation of China (82171818).

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Authors and Affiliations

  1. New Cornerstone Science Laboratory, Tsinghua-Peking Joint Center for Life Sciences, School of Basic Medical Sciences, Tsinghua University, Beijing, 100084, China

    Yu Li & Gong Cheng

  2. Laboratory of Advanced Biotechnology, Beijing Institute of Biotechnology, Beijing, 100071, China

    Yu Li, Xiaofan Zhao, Jiaqi Tang, Mengran Yi, Xiaodong Zai, Jun Zhang, Yilong Yang & Junjie Xu

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  1. Yu Li
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Contributions

J.J.X., Y.L.Y., and G.C. conceptualized the project. Y.L., X.F.Z., and J.Q.T. performed experiments and collected data. Y.L., Y.L.Y., M.R.Y., X.D.Z., and J.Z. analyzed and visualized the data. J.J.X. and Y.L.Y. supervised, directed, and managed the study. Y.L. and Y.L.Y. wrote the manuscript. All authors reviewed and edited the manuscript.

Corresponding authors

Correspondence to Gong Cheng, Yilong Yang or Junjie Xu.

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Ethics Animal housing and experimental procedures were conducted according to the Animal Care and Welfare Ethics Committee of Beijing Institute of Biotechnology.

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Supplementary Material 1: Supplementary Information includes additional file 1: Figure S1-S7.

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Li, Y., Zhao, X., Tang, J. et al. Endogenous capsid-forming protein ARC for self-assembling nanoparticle vaccines. J Nanobiotechnol 22, 513 (2024). https://doi.org/10.1186/s12951-024-02767-z

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Journal of Nanobiotechnology

ISSN: 1477-3155

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